For cancer cells to metastasize, they need to squeeze between other cells and through confined spaces in the extracellular matrix. Migration of cancer cells in tumors has been attributed to actin polymerization and myosin II-dependent contractility, based on studies of cell movement in unrestricted 2D environments. However, a recent publication made the potentially paradigm-shifting discovery that water permeation, not actin-myosin contractility, drives cell migration through physically confined, 3D microenvironments in vitro. The water permeation mechanism of cell migration, referred to as an osmotic engine, requires a polarized distribution of aquaporin and Na+/H+ pumps in the cell membrane that creates a net inflow and outflow of water and ions at the leading and trailing edges, respectively. Using microfluidic devices, this research will test the hypothesis that disrupting the osmotic engine will block migration of cancer cells in confined geometries. We also will investigate the hypothesis that disrupting the osmotic engine in malignant cells will abrogate spontaneous metastasis in mouse models of breast cancer, providing the first in vivo test of the osmotic engine as a driving force in cancer progression. If we show the osmotic engine to be a critical mechanism for cell migration in vivo, the results could drive new drug development to target this process. To test and short-circuit the osmotic engine in cancer cells, we will capitalize on our recent discovery of an approach to disrupt osmotic regulation of cells in response to mechanical stress. We have successfully expressed a bacterial mechanosensitive channel of large conductance (MscL) in the plasma membrane of mammalian cells and shown that this non-selective channel is activated by cytoskeletal tension. Since cell migration in confined space imposes significant cell deformation and increases tension of a cell, we hypothesize that MscL activation will negate the effects of the osmotic engine and block cell migration via two potential mechanisms: 1) MscL activation at the plasma membrane will abrogate polarized water permeation at the leading/trailing edges of migrating cancer cells; and 2) osmotic effects from MscL expression/activation on nuclear membrane that could alter stiffness of the nucleus, which has been shown to be the rate-limiting step during 3D cell migration in confined spaces. The PI's previous work suggests that MscL expression will introduce new mechanosensitive functions to living cells, providing a novel approach to investigate and potentially interrupt functions of the osmotic engine. This exploratory/developmental idea opens a completely unexplored arena in cancer biology that could provide new mechanistic understanding of cell migration in confined spaces present in tumor environments. The Specific Aims of this collaborative R21 research thus are: (Aim 1) Test the hypothesis that MscL expression will block migration of breast cancer cells in engineered confined microenvironments; (Aim 2) Test the hypothesis that expression of MscL will block spontaneous metastasis of breast cancer cells in a mouse model.